A Cardiac Troponin T Biosensor Based on Aptamer Self-assembling on Gold

In this study, a sensitive and accurate aptasensor was designed for early detection of myocardial infarction through the determination of troponin T (TnT). The successful immobilization of a specific aptamer sequence on the surface of gold that had a high affinity toward TnT was accomplished. TnT was electrochemically quantified. The results indicated that the aptasensor detected TnT in a range of 0.05-5 ng mL, and with a detection limit of 0.01 ng/mL. The performance of the aptasensor was investigated by analyzing 99 human serum samples. Both diagnostic specificity and sensitivity of the aptasensor were found to be 95%. The use of the designed aptamer-based biosensor could be an essential achievement in health policy, preventing deaths caused by myocardial infarction, and reducing patients with heart failure. The extensive use of this aptamer-based biosensor can also reduce costs, enhance speed, and improve accuracy in the diagnosis of TnT as an important myocardial infarction biomarker.

Since even mild myocyte damages lead to an increase in the level of cardiac biomarkers (9), using these biomarkers is very useful in the rapid diagnosis of myocardial infarction, and reduction of its complications. The most critical and specific biomarker for the rapid detection of myocardial infarction is troponin (10). Troponin is a protein that controls the relationship between actin and myosin, which plays an essential role in contractions of various muscles, including the heart muscle (myocard). This protein is a complex that contains three subunits consisting of troponin C (TnC), troponin T (TnT), and troponin I (TnI) (11).
Normally, the troponin level in the bloodstream is insignificant or indistinguishable. However, when the myocard is harmed, troponin is released into the bloodstream, and its concentration in the blood will increase; in this case, the more the damage to the myocard, the higher the blood troponin concentration will be (12). So far, most of the laboratory diagnostic methods for TnT assessment have been based on immunological assays (13,14).
Biosensors are promising tools in clinical and biomedical researches. They are potential alternatives for the routine bioanalysis methods and systems because of their simplicity and ability to analyze complex matrices. Biosensors for biomarkers detection are alternative tools for disease diagnostics with high sensitivity and specificity (15)(16)(17). For biomarker detection, the selection of the biosensor's bioreceptor is the troublesome primary step.
Using aptamers in the design of biosensors (aptasensors) has led to an increment in the specificity of the biosensors to capture the biomolecules (20). Aptasensors have relatively low cost and high binding affinity, and provide rapid diagnostic performance with high reproducibility (15,17,19). Compared to antibodies, aptamers have the advantage of easy production and biostability, and provide high selective biosensors applicable in cellular studies (21). Employment of aptamers in electrochemical biosensors has considerable attention because the combination of aptamer selectivity and electrochemical detection sensitivity makes the electrochemical aptasensors attractive tools for biological sample analysis (16)(17)(18)(19). Therefore, more studies on electrochemical aptasensors are recommended.
Up to now, some electrochemical biosensors have been designed for the detection of TnT, and most of them have been based on immunological methods using antibodies (22)(23)(24)(25)(26)(27)(28). In this research, a specific aptamer with a high affinity against TnT was used to fabricate an aptasensor to quantify TnT in biological samples.

Preparation of the TnT aptasensor
The polishing surface of the gold disk electrode was followed on a pad inoculated with 0.05 m-alumina powder and lubricated with water.
Polishing was continued till a mirror-face surface was obtained. To remove the alumina particles, the gold disk electrode was immersed in a 1:3 water/ethanol mixture, and sonicated in an The aptasensor was washed with deionized water, and was ready for use.

Detection of TnT
The aptasensor binding time for capturing the TnT molecules was followed at 37 C. All electrochemical measurements were performed using a -Autolab potentiostat/ galvanostat equipped with GPES 4.9 software (the Netherlands). A three-electrode system was used where a gold disk electrode, a platinum rod, and an Ag/AgCl, 3 M KCl electrode were applied as working, counter and reference electrodes, respectively. This attempt was evaluated using a

Data analysis
Data analysis was performed using GPES software version 4.9 and Excel software (2010).

Aptamer characterization and optimization of the OCP
Aptamers bind and recognize the protein targets through the secondary structure, folding, and 3D shapes of the aptamers as well as specific binding sites and different types of non-covalent attractions. There is a need for high avidity, affinity, specificity, and selectivity for the binding of aptamer-protein for an ultimate specific and sensitive biosensing. The secondary structure of the aptamer is shown in Figure 1.

Optimization of TnT binding time
For evaluation of the optimized time of TnT binding with the aptasensor, 0.5 ng/mL TnT was exposed at various times at 37 C. DPVs recorded before and after TnT incubation with the aptasensor at different binding times are shown in Figure 3.

TnT aptamer-based biosensor design and detection performance evaluation
The peak in the voltammogram is due to the redox transition of the marker (ferro/ferricyanide), and the peak current was decreased upon prolonging the TnT binding. The marker had a certain approachability to the aptasensor surface that was determined by the repulsion forces between the negatively charged aptamer and marker. On the other hand, TnT bears a net negative charge arising from its deprotonation in the working

Real matrix
Ref.

Reproducibility of the TnT aptasensor
In order to investigate the reproducibility of the TnT aptasensor fabrication, it was fabricated 6 times, and the related DPV for each time of fabrication was separately recorded. As shown in Figure 6, the current change in the voltammograms was very small, with a relative standard deviation (RSD) of 3.1%. This confirmed the regeneration ability of the aptasensor.

Repeatability of the TnT aptasensor
The repeatability of the TnT aptasensor to detect TnT was inferred by three independent measurements of TnT in one (an intra-day assay) or 3 days (an inter-day assay). The results showed RSD values lower than 4%. Besides, three determinations of 0.5 ng/mLTnT with a single aptasensor showed a RSD value of 3.5%.

Evaluation of the TnT aptasensor regeneration
The regeneration behavior of the TnT aptasensor was evaluated. This competency test was followed by six times binding-unbinding of 0.5 ng/mL TnT with the aptasensor, and the recorded pairs of DPVs after these cycles are shown in

Evaluation of the TnT aptasensor stability
The stability of the aptasensor was investi-   Table 2. any aptasensor signal smaller than/equal to Y-10× is considered as positive, where Y is the The results depicted that the aptasensor could detect TnT in the serum samples with two falsepositive and two false-negative outcomes.
Accordingly, diagnostic sensitivity and specificity of the aptasensor were obtained as 95%.

Discussion
Given the importance of myocardial infarction is to quantify the heart biomarkers (8).
The relationship between damage to heart myocytes and increased levels of cardiac biomarkers has been